This invention relates to a plant gene specifying the enzyme acetyl Coenzyme A carboxylase (ACCase) and to plant genomes genetically transformed with the said gene. Particularly, but not exclusively, the invention relates to ACCase genes from plants of the Brassica species, especially Brassica napus (oilseed rape) and control of expression of the gene by Brassica plants which are genetically transformed with the gene or its antisense configuration.
Acetyl Coenzyme A carboxylase is one of the genes involved in the synthesis of oil by oil-producing crops such as oilseed rape. Variation of the expression of that gene leads to alteration in the quantity and/or quality of the oil produced.
An object of the invention is to provide a gene specifying ACCase in plants.
According to the present invention there are provided partial cDNAs specifying ACCase, isolated from seed of Brassica napus, having the nucleotide sequences set forth in FIGS. 6 and 12, and variations thereof permitted by the degeneracy of the genetic code.
The invention further provides the partial cDNA, isolated from wheat germ, having the nucleotide sequence set forth in FIG. 4, and variants thereof permitted by the degeneracy of the genetic code.
Also provided by this invention is the full length genomic DNA specifying ACCase from Arabidopsis thaliana having the nucleotide sequence set forth in FIG. 8, and variants thereof permitted by the degeneracy of the genetic code.
The invention further provides the following clones, inserted in Escherichia coli, strain DHxcex1 hosts, which have been deposited with the National Collection of Industrial and Marine Bacteria, 23 St. Machar Road, Aberdeen, AB2 1RY, United Kingdom, on Mar. 25, 1993, under the provisions of the Budapest Treaty on the Deposit of Microorganisms for Patent Purposes, details of which are as follows:
1. Plasmid pK111, Accession No. NCIB 40553
2. Plasmid pKLU81, Accession No.NCIB 40554
3. Plasmid pRS1, Accession No. NCIB 40555
The present invention also provides genetically transformed plants, plant cells and plant parts, containing a DNA of the invention or fragment thereof in sense orientation or a complete or partial sense or antisense variant thereof.
It is preferred that the plant be of a species which produces substantial quantities of oil, rather than starch. Such plant species are well known and are simply referred to as xe2x80x9coil-seedxe2x80x9d crops and include, oilseed rape, canola, soya and sunflower. Methods for the genetic transformation of many oil crops are known; for example, transformation by Agrobacterium tumefaciens methods are suitable for most. Such methods are well-described in the literature and well-known and extensively practised in the art.
In our International Patent Application Number WO 92/19747, published on Nov. 12, 1992, we describe the biosynthesis of polyhydroxybutyrate from the substrate, acetyl-CoA. This activity involves three enzyme-catalysed steps. The three enzymes involved are xcex2-ketothiolase, NADP linked acetoacetyl-CoA reductase, and polyhydroxybutyrate synthase, the genes for which have been cloned from Alcaligenes eutrophus (Schubert et al, 1988, J Bacteriol, 170). In our international application we describe the cloning of these three gene into oil-synthesising plants.
However, the synthesis of fatty acids which are the building blocks of plant oils utilise the substrate acetyl Coenzyme A which is the same substrate required by the polyhydroxyalkanoate genes. By virtue of the present invention we provide means for down-regulating the fatty acid synthesis by inhibiting ACCase thereby leaving the acetyl CoA available for conversion to polyhydroxyalkanoates.
Methods for the regulation of gene expression are well-known in the art. Two principal methods are commonly employed, these being referred to loosely as xe2x80x9csensexe2x80x9d and xe2x80x9cantisensexe2x80x9d regulation. In antisense regulation a gene construct is assembled which, when inserted into a plant cell, results in expression of a messenger RNA which is of complementary sequence to the messenger produced by a target gene. The theory is that the complementary RNA sequences form a duplex thereby inhibiting translation to protein. The complementary sequence may be equivalent in length to the whole sequence of the target gene but a fragment is usually sufficient and is more convenient to handle. In sense regulation a copy of the target gene is inserted into the plant genome. Again this may be a full length or partial sequence. A range of phenotypes is obtained from which individuals in which the expression of the protein encoded by the target gene is inhibited may be identified and isolated as may individuals where expression of the gene product is increased. Sense regulation using partial sequences tends to favour inhibition. The mechanism is not well understood. Reference is made to European Patent Application No. 140,308 and U.S. Pat. No. 5,107,065 which are both concerned with antisense regulation and International Patent Application No. WO 90/12084 which describes sense regulation. The invention permits the following genetic modifications to be effected:
1. The clones of the invention may be used to probe plant DNA (genomic or cDNA libraries) to obtain homologous sequences. These may be truncated or full length cDNAs or genomic DNAs for ACCase genes from, for example, wheat, or oil crops such as rape, canola, soya, sunflower, maize, oil palm and coconut.
2. Partial cDNAs of rape seed ACCase may be used in conjunction with a plant-recognised promoter to create an expression cassette (partial sense or antisense) for use in transforming rape plants to down-regulate production of the ACCase enzyme. This will give plants with a lower oil content or oil of altered quality. The same cassette can be used to down-regulate the production of ACCase enzyme in other plants of the Brassica species. cDNAs isolated from other crops can be used to create expression cassettes (partial, sense or antisense) for use in transformation of these crops in order to modify the oil content.
Down-regulation of oil synthesis (in rape or other oil crops) can be used to divert the substrate, acetyl Coenzyme A, into synthesis of alternative storage materials such as starch, protein, or novel polymers introduced by genetic modification, for example polyhydroxyalkanoates.
3. Full length clones of rape or Arabidopsis ACCase DNA can be used to create expression cassettes, either with powerful promoters, or by inserting extra gene copies, to promote over-expression of ACCase in rape or other oil crops, leading to plants with enhanced oil content in the seed. The ACCase DNA may also be put under the control of a seed-specific promoter such as the napin promoter, which has a different window of expression from the ACCase promoter during seed development. In this way the period over which ACCase is expressed in the developing seed is extended, and the oil content of the seeds increased.
4. Genomic DNAs of rape ACCase can be used to recover the promoter of the ACCase gene. This promoter can be used to generate RNA in a tissue-specific and developmentally regulated fashion. The promoter so generated may promote the expression of ACCase, or it may control the expression of a gene construct placed after it (for example the structural gene of a different enzyme) which will then be expressed specifically in the developing seed.
5. The full length cDNA and genomic DNA of rape or Arabidopsis ACCase contain a sequence between the translation start site and the N-terminal sequence of the mature protein, known as a xe2x80x9ctransit peptidexe2x80x9d sequence. This directs the gene product to the plastids and is cleaved off during import of the protein into the plastids. This transit peptide sequence may be used in gene fusions to direct different gene products to the plastids.
6. Monocotyledonous plants, such as wheat, barley, maize and rice, are normally sensitive to the aryloxyphenoxy-propionate and alkylketone herbicides to which the dicotyledonous plants are normally resistant. Monocots with resistance to these herbicides may be created by:
(a) transforming ACCase from a dicotyledonous species such as rape and Arabidopsis, into the monocot genome;
(b) overexpression of the ACCase in a monocot; or,
(c) mutagenesis of ACCase and insertion of the mutant gene into a monocot.
7. It is believed that ACCase activity exists in both the plastid and the cytosol. Partial cDNAs of rape seed ACCase of this invention may be used in conjunctipon with a plant-recognised promoter to create an expression cassette (partial sense or antisense) for use in transforming plants to down-regulate production of the cytosolic ACCase. This will alter oil quality by inhibiting production of long chain fatty acids) chain length greater than about C18).
8. A second plastid form of ACCase has been identified in plants. This ACCase is composed of dissociable sub-units for transcarboxylase, biotin carrier protein (BCP) and biotin carboxylase (BC). The transcarboxylase gene is encoded by the chloroplast genome; BCP and BC are nuclear encoded. Sequence homology between the cDNAs of the invention and the BCP and BC may be used to isolate BCP and BC. Sense and antisense constructs may be raised against BCP and BC in order to effect down-regulation of these genes.
9. The cDNAs of the invention may themselves have sufficient homology with the BCP and BC genes to be used directly for the down-regulation of these genes.
We have prepared a poly dT primed cDNA library from developing rape seed and have obtained another from developing wheat embryo. These libraries have been probed with DNA fragments isolated earlier from a partial length maize leaf ACCase DNA (pA3) and partial length cDNA clones specifying rape seed ACCase (pRS1) and wheat germ ACCase (pK111) have thereby been selected and sequenced.
A DNA fragment isolated from the partial length rape ACCase DNA was then used to probe a genomic DNA library prepared from Arabidopsis thaliana and a full length Arabidopsis genomic DNA selected and sequenced.
The sequence of the Arabidopsis genomic DNA was used to generate specific probes by PCR. These were used to screen a random primed cDNA library from rape seed and two further rape ACCase partial cDNAs were thus isolated.
The full length Arabidopsis ACCase genomic DNA may then be used to probe a genomic library from rape and the full length rape ACCase genomic DNA selected and sequenced.
That the clones were indeed of ACCase genes was confirmed as follows:
The deduced amino acid sequence for wheat ACCase cDNA shows complete homology in four regions of sequence to the amino acid sequences obtained from four peptides isolated from the ACCase enzyme purified from wheat embryo. The deduced amino acid sequence shows high homology with both the rat and chicken ACCase genes. High homology at the amino acid level with maize leaf ACCase was found, with two sections of 48 amino acids completely conserved.
The deduced amino acid sequence from the rape seed partial cDNA (pRS1) sequence shows high homology to the sequences of the maize leaf cDNA and the chicken, rat, yeast and algal ACCase genes.
The deduced amino acid sequence from the Arabidopsis genomic DNA shows high homology with the rat, chicken and yeats ACCase genes. High homology with the amino acid sequence of the rape seed ACCase partial cDNA (pRS1) was found, with one section of 48 amino acids almost completely conserved.